Mechanism of integrin activation by talin and its cooperation with kindlin

Talin-induced integrin binding to extracellular matrix ligands (integrin activation) is the key step to trigger many fundamental cellular processes including cell adhesion, cell migration, and spreading. Talin is widely known to use its N-terminal head domain (talin-H) to bind and activate integrin, but how talin-H operates in the context of full-length talin and its surrounding remains unknown. Here we show that while being capable of inducing integrin activation, talin-H alone exhibits unexpectedly low potency versus a constitutively activated full-length talin. We find that the large C-terminal rod domain of talin (talin-R), which otherwise masks the integrin binding site on talin-H in inactive talin, dramatically enhances the talin-H potency by dimerizing activated talin and bridging it to the integrin co-activator kindlin-2 via the adaptor protein paxillin. These data provide crucial insight into the mechanism of talin and its cooperation with kindlin to promote potent integrin activation, cell adhesion, and signaling.


Major comments that would significantly improve the study include: In figure 1 what happens to cell adhesion, spreading and integrin activation of beta 1 integrin in the double null fibroblasts with the Tln TM mutants. I would also recommend making the label for this mutant something else as TM is also transmembrane domain.
Similarly these experiments should also be preformed in figure 3 and should be shown ass primary figures with the CHO cell data being supplemental. One acknowledges that it is excessively burdensome to do everything in cells where there is no expression of endogenous proteins, however this needs to be done for the major experiments as it is clear that the CHO system is artefactual and should not be the primary cell system used.
The discussion should be adapted so that the major points of the manuscript are easier to understand. If the authors perform the experiments above then the paradigms discussed in this paper would generalizable to both the b1 and b3 integrins and it may be considered a mechanism that works across most integrins.
How does this activation step intersect with talin-mediated FA formation? It would be nice to see an expanded discussion of Talin-mediated FA formation to "speculate" how talin, kindlin, and paxillian might assemble more than 2 integrins. Current discussion seems limited to two integrins for simplicity sake, yet significance of these finds quickly expands if we can see how it amplifies FA assembly. Can authors conclude how these mechanisms may limit FA assembly, i.e. can you speculate which components are essential, i.e. rate limiting? by regulating the binding of the integrin to multivalent ligands.

The main weakness of this work is the NMR-based structural characterization of the interaction between various talin-R fragments and paxillin protein.
There are a number of problems that are listened below 1) The authors report in the manuscript a quantitative description of the talin-R fragments and paxillin interactions by SPR measurements indicating that talin-R1-R4 is the principal region involved in the paxillin binding. However, they report that also talin-R9-R12 as well as talin-H display binding to paxillin. Overall, they find 7 subdomains including talin-F3, talin-R2, R3, R4, R8, R11 and R13. After that, they investigated the paxillin/talin-R fragment by acquiring 1H-15N Heteronuclear Single Quantum Coherence (HSQC) spectra focusing the attention on the 1-160 region of the paxillin 1-160 region. First, the material and methods section regarding the experimental NMR procedures (i.e acquisition and processing parameters; molar ratio between the two partners) is completely absent. Additionally, how did the author assign the chemical shifts? Did the authors use previously assigned chemical shifts? If a novel chemical shifts assignment has been performed the authors have to deposit the shifts in the Biomolecular Resonance Bank (BMRB) and report the BMRB ID code in the manuscript. Yet, the chemical shifts analysis reported for each domain is inappropriate (table S1). NMR is a powerful technique to describe per-residue conformational and dynamics features. Therefore, the authors have to i) include in the material and methods a detailed description of the procedure used to analyze the chemical shifts describing the used equation; ii) the plots showing the per-residue chemical shift perturbations for each of the talin-R fragments. It is not clear how the authors classified the data reported in the table S1. In all 1H-15N HSQC spectra illustrated in figures the chemical shifts assignment needs to be reported. 2) Moreover, if the talin-R1-R4 is the main portion in the regulation of the paxillin binding (KD= 171 nM) why didn't the authors use this portion for the NMR titration experiments? 3) Additionally, to provide a more complete description of the Talin/ paxillin, excluding that the isolated talin-R fragments may interact with the same paxillin binding site, the authors have to also see the binding from the paxillin side. In other word they have to perform NMR titration experiments with 15N-labeled 1-160 paxillin and unlabeled TalinR fragments or better talinTM (active form). These additional investigations will demonstrate their conclusion that the recognition process of talin-R by paxillin occurs via a multi-site binding mode as is already mention in the manuscript without any speculation. Moreover, to fully understand how the paxillin protein acts as link between talin and kindlin structural data regarding this supramolecular machinery is required. Therefore, the authors to address this latter point might perform the same NMR experiments adding also the kidlin protein. 4) Regarding the binding of the 15N-talin with two paxillin derived peptides the authors have to provide per-residue structural data reporting the chemical shift variations versus residue number. Additionally, they have to provide a more realistic structure and not only a simple structural model generated by sequence alignment and homology modelling ( Figure  4D). It is well known that the NMR is one of the most powerful technique for the structure determination. In this case, the author at least for one peptide have to calculate the threedimensional structure of the complex using CS (Chemical shifts)-or NOE-derived approach. 5) To confirm that talin-R2, R8 and R11 play important roles in the binding to paxillin the authors designed LD binding defective mutants named talin-R2 AKEE; R8 AKEE and R11 TKEE. In the figure 4C the 1H-15N HSQC spectra acquired for R2 WT and for the mutant talin-R2 AKEE with and without unlabeled 1-160 paxillin are reported. From the comparison of the pictures (figures 4C and 4E) it seems that the mutations induce significant structural variations (i.e the dispersion of resonances in both dimensions (proton and nitrogen) for the wt and the mutant are quite different). So far, the authors have to provide evidence that the mutations don't alter the 3D fold of the domain by comparing the chemical shift variations between wt/ R2 AKEE. The use of Ca chemical shifts would greatly help this structural analysis.

Minor points. 1) In the figures 4C and 4E the axis labels have to be corrected in 15N (ppm) and 1HN
2) In the figure S5 the authors in the figure legend report Free 15N,2H-talin-R2. Is the protein deuterated? If it is the case the protein is fully or partially deuterated?
In general, this work has the potential for high impact, as appropriate for publication in Nature Comm. However, the lack of high resolution structural data as well as the incomplete and/or inappropriate analysis of the NMR data, need to be addressed.

General remarks of the revision:
We greatly appreciate the reviewers' effort in evaluating our work. We have carefully gone over all the comments and made substantial effort to perform more experiments and revise our manuscript. The revision resulted in revised Fig  Because the change of the text is extensive, we highlighted major changes in the text in red. We hope the revision will be satisfactory since it addressed every question raised by the reviewers.
Reviewer #1 (Remarks to the Author): 1. For the SPR experiments, in the method section it is mentioned that each test was repeated, but I do not see the replicated data. How many times each test/concentration was repeated? The repeated response curves should be provided, and the mean and standard deviation (error range) of kinetic results based on the repeated measurements should be calculated. If the measurement were not repeated, please remove the repeat statement in method, and report the standard deviation of the fitting results (how well is the data fitting to the model).
Response: Thanks for the suggestion. All SPR experiments were indeed repeated at least twice with similar binding kinetics. The error range of each affinity is now provided in all revised SPR figures. We note that many experiments were repeated at different periods with different CM chips and non-identical immobilization conditions, resulting in somewhat different response curves that cannot be exactly aligned but the binding kinetics were similar so we provide one representative sensorgram for each figure with the error range indicated on all SPR figures.
2. Also for the SPR experiments, the paxillinFL was measured at 10 degree C (Fig. S5F), while the rest were measured at room temperature. The binding kinetics is a function of temperature based on thermodynamic principle. Have the authors take this temperature variation into consideration? The weaker KD of 350 nM between Talin-TM/paxillin could be due to this lower temperature measurement. I suggest adding this point to the discussion in page 13.

Response:
We did the paxillinFL binding to talin-TM at 10 degree C because we feel these full length proteins may be more stable at lower temperature during the whole assay. However, we just found the sample is fine for the SPR experiments at room temperature as long as we keep the uninjected samples at 10 degree C during the assay and the affinity is indeed stronger at higher temperature as reviewer predicted. To be consistent with other SPR data, we now provide the new data acquired at room temperature (now new Fig S7F).
Reviewer #2 (Remarks to the Author): 1. General:… Experimentally the major issue is the use of an artificial alphaiib beta3 integrin system in a CHO cell. This system is useful but also has some major problems especially with respect to understanding physiological relevance. These authors have talin double null cells so the manuscript would be significantly improved if they were used for more of the physiological readouts. In addition, the manuscript would benefit from significant revisions to extrapolate their findings more fully. Further, the details provided explaining the critical first steps of focal adhesion formation are arguably the most significant contribution of this paper and could be showcased more efficiently.

Response:
We thank the reviewer for the positive evaluation of our study and helpful comments to further improve it. We also appreciate the reviewer's valuable and critical thoughts on the CHO cell system. We agree that the CHO cell system has some drawbacks to precisely reflect the physiological processes but it is a useful system, as reviewer also agreed, to gain initial insight into the mechanism of integrin activation as demonstrated by many previous studies that were confirmed by subsequent in vivo analyses. Notably, it was this system that led to the discovery of talin as the key integrin activator (Calderwood et al. J. Biol. Chem, 274:28071-41999, cited >800 times), which is now widely accepted based on extensive in vivo studies. In our manuscript, the CHO cell system also led us discover the talin dimer-paxillin-kindlin (TPK) machinery in promoting integrin binding to multivalent ligands. We then used structural biology tools to determine the binding mode of the TPK complex and designed key mutation spots to disrupt the complex. These mutations allowed us to verify, again using the CHO cell system, the importance of this complex in regulating integrin activation. Finally, we used talin1/2 null fibroblasts to verify the physiological importance of the TPK complex in integrin activation and post-integrin activation events using the mutations we examined in CHO system. Thus, as you can see, these combined steps were highly integrated to effectively and definitively define the role of TPK in regulating the integrin activation.
Based on the reviewer´s suggestions, we made vigorous effort to perform more experiments in talin ½ null fibroblasts and then significantly revised the manuscript by putting our findings into a broader context (see also detailed responses to comments 2 and 3 respectively). We also extended our model shown in Figure 7C indicating that the TPK-induced integrin microclusters not only regulate integrin activation but further function as seeds to initiate focal adhesion formation.
Major comments that would significantly improve the study include: 2. In figure 1 what happens to cell adhesion, spreading and integrin activation of beta 1 integrin in the double null fibroblasts with the Tln TM mutants. I would also recommend making the label for this mutant something else as TM is also transmembrane domain.

Response:
Thanks for the suggestion. We have now functionally compared talin1/2 null fibroblasts expressing either talin-H (tlnH), talin-WT (tlnWT) or talin-TM (now labeled as tln-M3). Most importantly, while talin-H shows low potency in inducing cell adhesion as expected, expression of tln-WT and tln-M3 induced very similar cell adhesion and spreading, indicating that the retrovirally expressed talin-WT becomes activated by the endogenous activation machinery and does not require activating mutations. This is strikingly different from the CHO model and shows, as indicated by the reviewer, that the fibroblasts represent the more physiological cell system, but both data from CHO cell system and fibroblasts clearly indicate the importance of talin-R since talin-H clearly has low activity compared to either tln-WT or tln-M3. The new data are now shown in Figs 4A, 4B, and S3I. As tln-WT and tln-M3 both facilitate integrin mediated functions in a similar manner in talin1/2-null fibroblasts, the paxillin-binding defective talin mutant constructs, which were later expressed in talin-1/2null fibroblasts (Figs 4 and 6), were only done using the tln-WT backbone.
As mentioned above, we have changed our nomenclature for talin-TM as tln-M3.
3. Similarly these experiments should also be performed in figure 3 and should be shown ass primary figures with the CHO cell data being supplemental. One acknowledges that it is excessively burdensome to do everything in cells where there is no expression of endogenous proteins, however this needs to be done for the major experiments as it is clear that the CHO system is artefactual and should not be the primary cell system used.
Response: Please see our responses to point 1 (General) and point 2. We agree that the CHO cell systems has some drawbacks and therefore, as suggested by the reviewer, conducted a series of new experiments with our talin1/2 null fibroblasts. However, we still would like to keep the CHO cell data within Fig 3 as the PAC-1 binding assays also provide insights into the activity state of the aIIbb3 integrin, while the fibroblast assays address more general integrin-mediated adhesive activity. Thus, both models complement each other. Our new adhesion data on fibroblasts (new Fig 4) fully support the CHO data shown in Fig. 3. In particular, new Fig 4D shows that the synergistic function of talin and kindlin-2 on cell adhesion depends on the presence of the talin-R domain because talin-WT has much higher capacity to mediate cell adhesion than talin-H whereas in the absence of kindlin-2 talin-WT and talin-H exhibit no difference in mediating cell adhesion. We also specifically investigated the talin-paxillin-kindlin-2 complex by expressing a paxillin-binding deficient kindlin-2 mutant (GLKE) in kindlin-2 deficient fibroblasts, which resulted in reduced adhesion compared to cells that express kindlin-2 WT (new Fig 4F). Moreover, siRNA knockdown of paxillin, while reducing fibroblast adhesion in kindlin-2 WT expressing cells, did not further reduce adhesion when the paxillin-binding mutant kindlin-2 was expressed (new Fig 4F). These data confirmed our initial CHO data showing the importance of the paxillin-kindlin-2 interaction for integrin-multivalent ligand binding and cell adhesion. We also knocked out kindlin-2 in mouse embryonic MEF cells (MEFs) and observed significant adhesion defect in cells expressing the paxillin binding defective kindlin-2 mutant (GLKE) compared to the kindlin-2 WT (Fig S4A). We note that we also attempted to knock-out paxillin in MEFs but the cells displayed abnormal cell morphology and somehow showed dramatically reduced transfection efficiency of paxillin. We then switched to siRNA knock-down method to reduce endogenous paxillin in MEF cells and this approach allowed rather efficient paxillin transfection. Fig S4B shows that expression of paxillin WT but not K2 binding defective paxillin F577E mutant induced the ligand binding/cell adhesion, which cross-confirmed the K2 GLKE knock-in data in K2-KO cells (Fig S4A). Overall, our data clearly showed that disruption of paxillin-kindlin interaction in fibroblasts impairs ligand binding to integrin and cell adhesion. Overall, new Figs 4 and S4 provide strong evidence about the talin-paxillin-kindlin axis in regulating cell adhesion (see lines 219-251 in red for detailed description), which is fully consistent with the CHO cell data.
With regards to the analysis of focal adhesion formation, we would like to point out that our main goal in this study is to understand how integrin activation is regulated by talin-paxillinkindlin, which has not been reported before. Furthermore, the mutations that disrupt kindlin-2/paxillin interaction have already been shown to impair focal adhesion in our earlier study (ref 56). On the other hand, we showed in Fig 6 that disrupting talin/paxillin interaction impaired focal adhesion formation and cell spreading, which has never been reported so far. Together, all these data are consistent with our CHO cell-based data about the essential role of talin, paxillin, kindlin in integrin activation, cell adhesion and focal adhesion formation. 4. The discussion should be adapted so that the major points of the manuscript are easier to understand. If the authors perform the experiments above then the paradigms discussed in this paper would generalizable to both the b1 and b3 integrins and it may be considered a mechanism that works across most integrins.
Response: Almost the entire discussion section has been rewritten (in red) to better and more clearly reflect our points. As suggested by the reviewer, we also proposed that the mechanism on b1 and b3 integrins derived from this manuscript may be operative on other integrins. 5. How does this activation step intersect with talin-mediated FA formation? It would be nice to see an expanded discussion of Talin-mediated FA formation to "speculate" how talin, kindlin, and paxillin might assemble more than 2 integrins. Current discussion seems limited to two integrins for simplicity sake, yet significance of these finds quickly expands if we can see how it amplifies FA assembly. Can authors conclude how these mechanisms may limit FA assembly, i.e. can you speculate which components are essential, i.e. rate limiting?
Response: See the revised discussion and also revised Fig 7C where we show that integrin microclusters induced by the talin/paxillin/kindlin complex formation not only promotes the integrin binding to multivalent ligands but also serve as seeds for the formation of focal adhesions. We feel that the expression level of paxillin, which is lower than talin and kindlin, may be critical for regulating the ternary complex assembly and determining the growth of FAs (see lines 488-493).
6. Is it still correct to distinguish between "micro" clustering and ECM-induced FA formation. It seems data herein is defining the first steps in "macro" clustering that are not necessarily ECM induced. This is found in line 424-28.
Response: This is an important point. Our description of "micro" clusterings refers to their early occurrence and small scale, in comparison to the "macro" clusterings (FAs) that are normally visible with standard microscopes and require at least one hour to form. To further distinguish the two, we indicated in the discussion (lines 483-487) that microcluster formation is mostly triggered and interlinked via intracellular signaling and adaptor proteins, which prepares cells for efficient cell adhesion (not necessarily binding to ligands). But as an early and critical event, it will also impact following events, for example, cell adhesion and spreading, while these experiments can only be conducted on ECM ligand coated surfaces.
More minor comments include: 7. Line 110: a supplementary figure to support the logic of the chosen talin mutations would be of benefit to the reader. 11. Line 445-48: consider rephasing, logic of this sentence is difficult to follow.

Response:
The sentence has been rewritten.
Reviewer #3 (Remarks to the Author): The main weakness of this work is the NMR-based structural characterization of the interaction between various talin-R fragments and paxillin protein. There are a number of problems that are listened below 1) ... First, the material and methods section regarding the experimental NMR procedures (i.e acquisition and processing parameters; molar ratio between the two partners) is completely absent. Additionally, how did the author assign the chemical shifts? Did the authors use previously assigned chemical shifts? If a novel chemical shifts assignment has been performed the authors have to deposit the shifts in the Biomolecular Resonance Bank (BMRB) and report the BMRB ID code in the manuscript. Yet, the chemical shifts analysis reported for each domain is inappropriate (table S1). NMR is a powerful technique to describe per-residue conformational and dynamics features. Therefore, the authors have to i) include in the material and methods a detailed description of the procedure used to analyze the chemical shifts describing the used equation; ii) the plots showing the per-residue chemical shift perturbations for each of the talin-R fragments. It is not clear how the authors classified the data reported in the table S1. In all 1H-15N HSQC spectra illustrated in figures the chemical shifts assignment needs to be reported.

Response:
We apologize that our NMR method section was too brief. This section has been substantially expanded with more experimental details. The molar ratio between the two partners was actually provided in original figure legends of 4C, 4E, S4F, S6, and S7G (now 5C, 5E, S6, S8, S9B-C, S10A-E). We also mention this now in the method section so that readers will know where to find the information.
Regarding chemical shift assignment, we wish to clarify that our major goal of acquiring HSQC of all talin-R subdomains was to verify the binding results derived from pull-down experiments and more specifically define which subdomain is involved in binding to paxillin. HSQC spectra of each subdomain in the absence and presence of paxillin 1-160 as pair were sufficient to reveal the binding based on chemical shift change of the paired spectra so there is no need to know the chemical shift assignment. Using this approach, we found that R1, R5, R6, R7, R9, R10, R12 had no chemical shift changes (not shown) whereas six other subdomains including R2/R3/R4/R8/R11/R13 had chemical shift changes upon addition of paxillin 1-160. Among these six subdomains, R2/R8/R11 had much more significant chemical shift changes by paxillin (Figs 5C/5E, S5A-B) than R3 and R4 (included as new Fig S6C-D) and R13 had very tiny/almost negligible chemical shift changes by paxillin (new Fig S6E). Based on this observation, we classified R2/R8/R11 as main paxillin binding sites (++) and R3/R4/R13 as weak paxillin binding sites and summarized them in Table S1. The strengths of the R2/R8/R11 binding to paxillin 1-160 were further quantified later in our SPR experiments with the affinities ranging from 2 to 13 M (Figs S7A-7C). We revised the text to more clearly describe our approach (lines 281-289).
Since the chemical shifts of R2 and R8 have been published before (BMRB code 17350 and 19339, respectively), we followed the reviewer's suggestion to perform the chemical shift mapping analysis using the equation provided in the method section. New Figs S9B and C show the chemical shift perturbation patterns of R2 and R8 by paxillin 1-160. Since R8 was shown to bind paxillin LD homolog peptide DLC-LD (PDB 5FZT), we hypothesized that LD motifs (Fig S9A) in paxillin 1-160 play important roles in binding to the talin-R subdomains. Indeed, their chemical shift perturbation patterns by LD1 or LD2 (contained in paxillin 1-160) were similar to those of paxillin 1-160 (new Figs S9A-B). The binding results of talinR2/R8/R11 and their mutants to LD1 and LD2 are summarized in Table S2. New Figs S9D-E further show the perturbed LD binding surfaces on R2 and R8, which are similar, and the latter is fully consistent with the known crystal structure of talin-R8 in complex with DLC-LD peptide ( Fig S9F). Remarkably, key residues in the LD binding surface of R8, which is structurally similar to R2 and R11, are conserved (Figs S9F vs 5D and S9G), allowing us to design point mutations to disrupt these Paxillin 1-160 LD/talin-R subdomain interaction (Figs S5E-F, S11A-D, and see also Table S2). These data demonstrate that paxillin LD motifs play crucial role in binding to talin-R. We have revised the text to reflect these changes (lines 328-364).
2) Moreover, if the talin-R1-R4 is the main portion in the regulation of the paxillin binding (KD= 171 nM) why didn't the authors use this portion for the NMR titration experiments?
3) Additionally, to provide a more complete description of the Talin/paxillin, excluding that the isolated talin-R fragments may interact with the same paxillin binding site, the authors have to also see the binding from the paxillin side. In other word they have to perform NMR titration experiments with 15N-labeled 1-160 paxillin and unlabeled TalinR fragments or better talinTM (active form). These additional investigations will demonstrate their conclusion that the recognition process of talin-R by paxillin occurs via a multi-site binding mode as is already mention in the manuscript without any speculation. Moreover, to fully understand how the paxillin protein acts as link between talin and kindlin structural data regarding this supramolecular machinery is required. Therefore, the authors to address this latter point might perform the same NMR experiments adding also the kidlin protein.